Advance Journal of Food Science and Technology 1(1): 35-38, 2009
ISSN: 2042-4876
© M axwell Scientific Organization, 2009
Submitted Date: September 12, 2009
Accepted Date: October 12, 2009
Published Date: November 10, 2009
Microbiological Analysis of Pathogenic Organisms in Indigenous
Fermented Milk Products
S.B. K umbhar, J.S. G hosh and S.P. Sam udre
Department of Microbiology, Shivaji University, Kolhapur 416004, MS. INDIA.
Abstract: Milk is one of the m ost affordable source of many nutritions like proteins and vitamins. Many peop le
do not like to consume m ilk in its liquid form. T herefore, there are sev eral products which are mad e out m ilk
called as Dairy Products, which preserve the nutritive values of m ilk and mak es it easily acceptable to
consumers. One such class of products, is the fermented dairy products. In India these are named in vernacular
languag e as Da hi (lactic fermented an d curdled m ilk), Shrikhand with different flavours prepared from hung
curd, sugar and real fruit pulps, lassi w hich is nothing but a so rt of butter milk w ith slightly high m ilk solid
content and sweetened with sugar. How ever a large volume of these products are produced in unorganized
sector, unbranded, with little precautions of food safety and quality. This investigation is a factual
documentation of such a finding and sugg estions as to the methods needed to improve the safety and quality.
Key w ords: Dahi, shrikh and, lassi, micrococcus, escherichia and m ilk
INTRODUCTION
Food is the matter of health and a matter of trust also.
Traditionally for several centuries man has adopted
fermentation as a means of food preservation and also
found them to have nutrition al and therapeutic
advantages. Fermented milk products constitute a vital
component of the human diet in many regions of the
world. In the Indian sub-continent such products are also
classified as “indigenous milk products” like dahi (curd),
lassi, shrikhand etc. which are prom inent in peop les diet.
Indigenous milk products refer exclusively to dairy
products of a particular region or co untry. In the present
context, these are products of Indian origin.The
significance of milk in hum an nu trition in no w w ell
established as it is con sidered as the best, ideal and
com plete food for all age groups. Milk and dairy products
are an excellent source of calcium, phosphorus and
magnesium. These minerals in optimum ratio are present
in milk and are required for optimum growth and
maintenance of bones (Aneja et al., 2002 ). However, in
spite of this, milk can also serve not only as a potential
vehicle for transmission of some pathogens but also
allows these o rganisms to grow, multiply and produce
toxins. A variety of pathogenic organisms m ay ga in
access in to milk and milk products from different sources
and cause different types of food born illnesses. Milk and
milk products m ay carry toxic metabolites of different
organisms grow ing in it. Ingestion of such products,
contaminated with these metabolites, cause food
poisoning. On the other hand the ingestion of viable
patho genic bacteria along with the food product leads to
food borne infection. Some time these organisms undergo
lysis in the gastrointestinal tract and liberate toxic
substances from inside the cells which are detrimental to
the health of the consumers (Aneja et al., 2002). Recent
development regarding Quality and safety management
systems such as ISO and Hazard Analysis Critical Control
Point (HA CC P) has reduced such incidences. Indian
Standard Institution in their “Specification for fermented
milk products” (IS: 7035, 1973) has clearly defined the
acceptable quality of such products, which wou ld be safe
for consumption.
This study has been attempted to carry out a survey
of some loc al suppliers of such products within an area of
radius of 100 km.
MATERIALS AND METHODS
This studies were con ducted over a period of 24
months to see the difference in the quality in different
seasons. The period of study was June 2007 to May 2009.
Sampling of dairy products from market:
Representative samples of different dairy products like
Dahi, Shrikhand (various flavors) and lassi were picked
up from retail market at random depending on the volume
of stock w ith the retailer. If required, com posite samples
were made, of each prod uct from eac h retailer, in the
laboratory. The samples were then subjected to different
tests which w ere ph ysical like pH, temperature, color,
odor and feel of consistency. These were the subjected to
chemical tests like
Estimation of fats: The fat content of the prod ucts were
estimated by Gerbe r method (IS: 1224, 197 7) (Fig. 1).
Estimation of M oisture con tent: The moisture content
of all the products were estimated (IS: 11623, 1986)
(Fig. 2).
Correcponding Author: Dr. J. S. Ghosh, Dept of Microbiology, Shivaji University, Kolhapur 416004.Ph: +91 9850515620
35
Adv. J. Food Sci. Technol., 1(1): 35-38: 2009
Estimation of Titrable Acidity: The titrable acidity of all
samples are detected as per (IS: 11766, 1986), which is
the average results of 12 sam ples of each products
(Fig. 3).
Microbiological analysis: The samples were
homogenized in a blender using sterile physiological
saline (0 .85% NaCl solution).
Coliform count: The supernatant of the above mentioned
suspension was diluted 1:10 and was directly plated on
Violet Red Bile Agar (IS: 5404, 1995). The inoculated
plates of this medium were incubated 37ºC for 48 h
(Fig. 4). In case of suspected colonies, the sam ples were
then enriched in B rilliant G reen L actose Bile Broth
(BGLB) having dehydrated ox bile-2%, lactose-1%,
peptone-1% and 1 .3 mL of 1.0 % solution of brilliant
green in water, final pH 7.2. The tubes were incubated at
37ºC for 48 h . This growth in these tubes were then
inoculated on Hicrome E. coli agar (Hi Media L td.,
Bom bay). The plates were incubated at 30ºC for 4 h and
then at 44ºC for 18 h (Tab le 1).
Fig. 1: Fat content of the products examined. The standard
values are as per PFA-1954 (2009)
Micrococcus aureus: Since milk is very good nutrient for
Micrococcus aureus, the supernatant of the homogenized
samples were subjected to detection of this
microorganism, which is a potent exotoxin producer
(Draft ISO/DIS 6888, 1983). The isolates were checked
for coagulase positive nature using defibrinated plasma
(Table 2).
Fig. 2: Moisture of the different products. The standard values
shown in figure are as per PFA-1954 (2009)
Salmonella: Detection of Salm onella were carried out as
per the prescribed metho d of (IS: 5887, 1999).
Shigella: The presence or absence of Shigella spp. was
carried out as per (IS: 5887, 199 9).
Listeria monocytogenes: The de tection of Listeria
monocy togenes was don e as per (ISO 10560, 19 99).
Fig. 3: Titrable acidity of the different products. The standards
as shown in the figure are as per PFA-1954 (2009)
RESULTS AND DISCUSSION
It can be seen from the Fig. 1 that only in case for
shrikhand and fruit shrikhan d, there is a large unjustifiable
deviation from the standard, which often does more ha rm
than good.
It is only in case of shrikhand and amrakhand, that
titrabele acidity is below standard which might pose not
only a threat of spoilage by microorganisms, but allow
food poisoning organisms like Micrococcus aureus to
grow and make the product hazardous. (Fig. 3)
It may be noted from the F ig. 2 that Mo isture
contents of the products men tioned are w ell within limits.
How ever, since th ere are no standards for D ahi and Lassi,
these parame ters were not checked as there is no means of
com parison.
Fig. 4: Coliform count of different products. The standards
shown above are as per PFA-1954 (2009)
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Adv. J. Food Sci. Technol., 1(1): 35-38: 2009
Table 1: Microbiological analysis of Different Dahi samples
S. No.
Body &
En t E. co li /gm
Salmonella /gm
Tex ture
1.
Loose
ND
ND
2.
Firm
Present
ND
3.
Loose
ND
ND
4.
Loose
ND
ND
5.
Loose
ND
ND
6.
Loose
Present
ND
7.
Loose
Present
ND
8.
Firm
Present
ND
9.
Loose
ND
ND
10.
Firm
Present
ND
11.
Firm
ND
ND
12.
Loose
ND
ND
ND : Not Detected
Table 2: Microbiological analysis of Shrikhand samples
S. No.
En t E. coli /gm
Salmonella /gm
1.
ND
2.
ND
3.
Present
4.
ND
5.
Present
6.
ND
7.
ND
8.
ND
9.
Present
10.
ND
11.
Present
12.
ND
ND : Not Detected
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Shigella/gm
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Shigella /gm
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Table 3: Microbiological analysis of Shrikhand with mango pulp samples
Sr. No.
En t E. co li /gm
Salmonella /gm
Shigella /gm
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
ND : Not Detected
ND
ND
ND
ND
ND
Present
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
It is vivid from the results above that 3 out of 4
products shows high coliform count which is detrimental
to the safe ty of the product, from public health point of
view (Fig.4).
It can be seen from Table 1, that enteric E. coli was
present in 5 out of 12 samples and Micrococcus aureus is
present in 2 out of 12 samp les. This means that chances of
contracting an enteric disease through consumption of
such curds is nearly 50%
Shrikhand is a sweetened product made out of hung
curd, but the sugar is used as a flavouring agent rather
than reduc ing w ater activity. However, there are certain
spices like crushed cardamom seeds, the oleoresins of
which would inhibit the growth of many microorganisms
Micrococcus
aureus /gm
ND
ND
ND
ND
Present
Present
ND
ND
ND
ND
ND
ND
Micrococcus
aureus /gm
ND
Present
ND
ND
ND
ND
ND
ND
Present
ND
ND
ND
Micrococcus
aureus /gm
ND
ND
ND
ND
ND
Present
ND
ND
Present
ND
ND
ND
Liste ria
monocytogens /gm
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Liste ria
monocytogenes /gm
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Liste ria
monocytogenes /gm
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
and that is why it is observed from Table 2, that 4 out of
12 samples which were really prepared in unhygienic
conditions show s the presenc e of E. coli and 2 shows the
presence of Micrococcus aureus. Therefore, this product
has more chan ces of giving food poisoning cases as
Micrococcus aureus is a potent exo toxin p roducer.
This is a product like shrikhand. The only difference
is that it contains actual Alphonso mango pulp. Ag ain the
point that is to be noted from Table 3, is that, there is
presence of Micrococcus aureus which is a potent
exotoxin producer, capable of causing food poisoning.
In fruit shrikhand, pineapple, grape, apple and
pom egran ate was found to have been added shrikhand.
Table 4 show that 2 samples were found to be safe as only
one of it show ed the presence of E. coli.
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Adv. J. Food Sci. Technol., 1(1): 35-38: 2009
Table 4: Microbiological analysis of Fruit Shrikhand other than mango samples
Sr. No.
En t E.co li /gm
Salmonella /gm
Shigella /gm
1.
2.
ND : Not Detected
ND
Present
ND
ND
ND
ND
Table 5: Microbiological analysis of Lassi samples
Sr. No.
En t E.co li /ml
Salmonella /ml
Shigella /ml
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Present
Present
Present
Present
Present
ND
ND
ND
ND
Present
ND
Present
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Micrococcus
aureus /gm
ND
ND
Liste ria
monocytogenes /gm
ND
ND
Micrococcus
aureus /m
Present
Present
Present
Present
ND
ND
ND
ND
ND
ND
ND
ND
Liste ria
monocytogenes /ml
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND : Not Detected
This product is prepared from curd, which was mixed
with water to get certain pulp density. There is no
standard for the pulp density and due to this every
manufacturer would add any amount of water they like as
long as they the customers for their product. There is also
presence of sugar in this product. Table 5 show that out of
12 samples the re are about 7 sam ples co ntaining enteric
E. coli and 4 ou t of 12 con tained Micrococcus aureus.
Production of this is mo stly in the unorganized sector and
hence there is no brand that needs to be pu t nor there is
any need for a definite retail outlay to sell this prod uct.
All this factors make the manufacturer free to add any
water they like and these contam inants are from the water.
products should be educated in the minimum
requirements of good hygienic and sanitary conditions,
good manufacture practices and good storage conditions
which will reduce the risk of poor microbiological quality
and increase the safety of the products.
REFERENCES
Aneja, R.P., B.N. Mathur, R.C. Chandan and A.K.
Banerjee, 2002. Technology of Indian Milk Products;
A D airy Ind ia Pub lication, D elhi.
Draft ISO/DIS 6880, 1983. International Organization for
S t a n d a r d i z at i o n ( I S O ) , e n u m e r a t io n o f
Staphylococcus aureus.
IS: 7035, 1973. Specification for ferm ented milk
products. N ew Delhi.
IS: 1224, 1977. Part-II, Determination of Fat by Gerber
Method: Milk Prod ucts Indian Standards Institution,.
Manak Bhavan New D elhi.
IS: 11766, 1986/ISO: 6092, 1980. M ethod for
determination of titratable acidity in milk powder and
similar products (routine method ).
IS: 11623, 1986. Re-1997, Method for determination of
moisture content in milk powder and similar
products.
IS: 5404, 1995. Methods for drawing and handling of
food samples for microbiological analysis.
IS: 5887, 199 9. Part 3. Method s for detection of bacteria
respo nsible for food poisoning.
ISO 10560, 1999. Milk and milk products-Detection of
Listeria monocytoge nes. Bratislava. pp: 24.
PFA (Prevention of Food Adulteration Acts (India)-1954,
2009. Food Safety Laws, International Law Book
Publishers, N ew Delhi.
CONCLUSION
Mo st of the Indigenous fermented milk products are
prepared by traditional methods in the unorganized
industrial sector. Such methods often bring about
contamination of various microorganisms including
pathogens. To overcome these problems these traditional
methods are automated in different organized sectors
which shou ld hav e helped to improve the microbiological
quality of the products. However, due to high cost of
capital investment, a sizeable portion of these products are
still produced in the unorganized sectors, many of which
do not even have the necessary licenses to do so and
hence are not bound by any law of product safety. These
are, more than often, not branded and the volume of sales
are significantly high but localized regionally. This has
resulted in poo r microbiolog ical quality with respe ct to
presence of pathogens and spoilage organisms. Therefore,
If the taste and flavors of these tradional flavors is to be
maintained traditionally, the producers of these high risk
38